Progesterone attenuates temporomandibular joint inflammation through inhibition of NF-κB pathway in ovariectomized rats

www.nature.com/scientificreports OPEN Received: 30 August 2017 Accepted: 20 October 2017 Published: xx xx xxxx Progesterone attenuates temporomandibular joint inflammation through inhibition of NF-κB pathway in ovariectomized rats Xin-Tong Xue1,2, Xiao-Xing Kou1,2, Chen-Shuang Li1,2,4, Rui-Yun Bi3,5, Zhen Meng3,6, Xue-Dong Wang1,2, Yan-Heng Zhou1,2 & Ye-Hua Gan3 Sex hormones may contribute to the symptomatology of female-predominant temporomandibular disorders (TMDs) inflammatory pain. Pregnant women show less symptoms of TMDs than that of non-pregnant women. Whether progesterone (P4), one of the dominant sex hormones that regulates multiple biological functions, is involved in symptoms of TMDs remains to be explored. Freund’s complete adjuvant were used to induce joint inflammation. We evaluated the behavior-related and histologic effects of P4 and the expression of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 in the synovial membrane. Primary TMJ synoviocytes were treated with TNF-α or IL-1β with the combination of P4. Progesterone receptor antagonist RU-486 were further applied. We found that P4 replacement attenuated TMJ inflammation and the nociceptive responses in a dose-dependent manner in the ovariectomized rats. Correspondingly, P4 diminished the DNA-binding activity of NF-κB and the transcription of its target genes in a dose-dependent manner in the synovial membrane of TMJ. Furthermore, P4 treatment showed decreased mRNA expression of proinflammatory cytokines, and partially reversed TNF-α and IL-1β induced transcription of proinflammatory cytokines in the primary synoviocytes. Moreover, progesterone receptor antagonist RU-486 partially reversed the effects of P4 on NF-κB pathway. In conclusion, progesterone ameliorated TMJ inflammation through inhibition of NF-κB pathway. Temporomandibular disorders (TMDs) are approximately twice as prevalent (and more severe) in women than in men, similar to rheumatoid arthritis1. Sex hormones are reported to be involved in TMD pain1–3. Joint inflammation is believed to be a chief cause of pain in patients with TMD1,4,5. We and other groups have reported that estrogen aggravates TMJ inflammation or pain through the induction of proinflammatory cytokines in the synovial membrane6–11. However, it is still difficult to explain why women with TMD experience some amelioration of pain during pregnancy12, even though estrogen increasing throughout pregnancy. Some other hormones may also be involved in TMD pain. Similar to estrogen, progesterone also increase dramatically and steadily during pregnancy12,13. Whether progesterone attenuates to TMJ inflammation and pain remains to be explored. Progesterone is involved in inflammatory response and immune modulating13,14. Progesterone decreases interlukine (IL)-6-induced expression of gp130 in hybridoma cells 15 and inhibits the expression of matrix 1 Department of Orthodontics, Peking University School and Hospital of Stomatology, Beijing, China. 2Center for Craniofacial Stem Cell Research and Regeneration, Peking University School and Hospital of Stomatology, Beijing, China. 3Center for Temporomandibular Disorders and Orofacial Pain, Peking University School and Hospital of Stomatology, Beijing, China. 4Section of Orthodontics, Division of Growth and Development, School 8of Dentistry, University of California, Los Angeles, Los Angeles, California, USA. 5The Third Dental Center, Peking University School and Hospital of Stomatology, Beijing, China. 6Precision biomedical laboratory, Liaocheng People’s Hospital, Liaocheng, China. Xin-Tong Xue and Xiao-Xing Kou contributed equally to this work. Correspondence and requests for materials should be addressed to Y.-H.Z. (email: ) or Y.-H.G. (email: kqyehuagan@ bjmu.edu.cn) SCienTifiC REPOrTs | 7: 15334 | DOI:10.1038/s41598-017-15285-w 1 www.nature.com/scientificreports/ Figure 1. Progesterone attenuated CFA-induced TMJ mechanical hyperalgesia and inflammation. (A) Serum levels of progesterone in intact rats and progesterone-treated ovariectomized rats. Among the ovariectomized rats, serum levels of progesterone were lowest in 0 μg-P4 treated rats and increased in a dose-dependent manner with the increasing of injected progesterone. *P < 0.05 versus control and sham; #P < 0.05 versus 0 μg; &P < 0.05 versus 20 μg, by two-way analysis of variance (ANOVA). (B,C) Head withdrawal threshold before (B) and after (C) TMJ inflammation. Progesterone ameliorated the TMJ inflammation–induced decrease in the head withdrawal threshold. (D) Representative images of the TMJ regions in female rats. Small and large black frames indicate the original and the magnified areas, respectively. Following induction of inflammation for 24 hours, TMJs of the sham-OVX and the 0-μg-P4 group showed typical features of synovitis. Compared with the 0-μg-P4 group, the proliferation of synoviocytes was less than that in 350-μg-P4 group. In 700-μg-P4 group, the synovial lining layer was thin and infiltration of mononuclear cells around the synovial membrane was not evident. (E) Progesterone attenuated CFA-induced TMJ inflammation. Scores for TMJ inflammation decreased with increasing doses of P4. *P < 0.05 versus control; #P < 0.05 versus sham; &P < 0.05 versus 0 μg, by two-way ANOVA. matalloproteinases in cultured human synoviocytes16. Progesterone attenuates rat paw hyperalgesia induced by complete Fruend’s adjuvant. More importantly, progesterone treated mice show decreased arthritis scores and decreased expression of IFN-γ17. However, the mechanism underling antiinflammatory effect of progesterone in joint inflammation remains largely unknown. NF-κB pathway is crucial in the modulation of inflammatory response17. Various stimuli and proinflammatory cytokines lead to the nuclear translocation of NF-κB and subsequently promoting transcription of IL-1β, IL-6, and tumor necrosis factor-α (TNF-α)18, which appear to be the major proinflammatory cytokines involved in TMJ pathology19. Previously, we reported that estrogen-enhanced activation of NF-κB aggravates to TMJ inflammation, and blocking NF-κB attenuates inflammation and pain of TMJ6. It has been demonstrated that progesterone decreases cyclooxygenase-2 (COX-2) expression in myometrial cells by inhibits the activity of NF-κB20, and a negative interaction exists between the progesterone receptor and NF-κB p65 in both COS-1 and HeLa cells21. Whether progesterone inhibits activation of NF-κB in inflamed joint remains to be examined. In this study, we explored whether progesterone could attenuate TMJ inflammation and pain, and whether progesterone could inhibit NF-κB activity and NF-κB-modulated transcription of proinflammatory genes in the synovial membrane of inflamed TMJs in ovariectomized rats. Materials and Methods Animals. Adult female Sprague-Dawley (SD) rats were purchased from Vital River Laboratory (Beijing Vital River Laboratory Animal Technology Co., Ltd., China). The experimental protocols (...truncated)